The PPAR-γ Agonist Pioglitazone Modulates Proliferation and Migration in HUVEC, HAOSMC and Human Arteriovenous Fistula-Derived Cells.

The failure of arteriovenous fistulas (AVFs) following intimal hyperplasia (IH) increases morbidity and mortality rates in patients undergoing hemodialysis for chronic kidney disease. The peroxisome-proliferator associated receptor (PPAR-γ) may be a therapeutic target in IH regulation. In the present study, we investigated PPAR-γ expression and tested the effect of pioglitazone, a PPAR-γ agonist, in different cell types involved in IH. As cell models, we used Human Endothelial Umbilical Vein Cells (HUVEC), Human Aortic Smooth Muscle Cells (HAOSMC), and AVF cells (AVFCs) isolated from (i) normal veins collected at the first AVF establishment (T0), and (ii) failed AVF with IH (T1). PPAR-γ was downregulated in AVF T1 tissues and cells, in comparison to T0 group. HUVEC, HAOSMC, and AVFC (T0 and T1) proliferation and migration were analyzed after pioglitazone administration, alone or in combination with the PPAR-γ inhibitor, GW9662. Pioglitazone negatively regulated HUVEC and HAOSMC proliferation and migration. The effect was antagonized by GW9662. These data were confirmed in AVFCs T1, where pioglitazone induced PPAR-γ expression and downregulated the invasive genes SLUG, MMP-9, and VIMENTIN. In summary, PPAR-γ modulation may represent a promising strategy to reduce the AVF failure risk by modulating cell proliferation and migration.

Impact of Freeze-Drying on Cord Blood (CB), Serum (S), and Platelet-Rich Plasma (CB-PRP) Preparations on Growth Factor Content and In Vitro Cell Wound Healing.

Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option.

Biomarkers in Tears and Ocular Surface: A Window for Neurodegenerative Diseases.

OBJECTIVES: The purpose of this review is to briefly outline current scientific evidence on the potential role of tear analysis and ocular surface evaluation in diagnosis and monitoring of neurodegenerative diseases, especially Alzheimer disease, Parkinson disease, and glaucoma. METHODS: A systematic computerized search in the electronic databases PubMed, MEDLINE, and the Cochrane Collaborations was conducted to find eligible articles which their main topic was to investigate the tear and ocular surface in neurodegenerative diseases. After a first screening of titles and abstracts and a full-text review, 26 articles met the inclusion criteria (1 about the neurodegenerative diseases, 3 about the Alzheimer disease, 11 about the Parkinson disease, 11 about glaucoma, and 1 about amyotrophic lateral sclerosis). RESULTS: The ocular surface picture seems to be altered in the setting of neurodegenerative diseases with specific characteristics according to each disease. They seem to be associated with reduced corneal sensitivity and abnormal tear function, and each one presents the expression of specific biomarkers in tears. CONCLUSIONS: The study of tears and ocular surface appears to be a new and noninvasive promising way to assist in the diagnosis and monitoring of neurodegenerative diseases.

There is a Role in Detection of SARS-CoV-2 in Conjunctiva and Tears: a comprehensive review.

Data on the involvement of the ocular surface and its relationship with Coronavirus disease 2019 (COVID- 19) are still minimal and not univocal. The respiratory tract is the structure most affected by COVID-19, and the serious form of the disease is characterized by severe pneumonia, acute respiratory distress syndrome and hypercoagulation. However, accumulating evidence shows that other organs could be reached by the virus, thus causing further comorbidities. To date, the exact route/routes of transmission of COVID-19 are still unclear. The respiratory tract is probably not the only route of transmission for this viral infection and some authors have also speculated that COVID-19 droplets, or infected hands, could contaminate the conjunctiva, which could therefore represent the initial site of an infection spread. Theoretically, the role of the ocular surface, a biological site still relatively unexplored, appears scientifically relevant in understanding the Severe Acute Respiratory Syndrome - Coronavirus - 2 (SARS-CoV-2) infection. The purpose of this paper is to summarize the current literature in order to elucidate the potential role of tear and conjunctival sampling to detect SARS-CoV-2 for the diagnosis of COVID-19 and to monitor patients during follow-up.

Dry Eye Disease and Tear Cytokine Levels-A Meta-Analysis.

Background-It is recognized that inflammation is an underlying cause of dry eye disease (DED), with cytokine release involved. We systematically reviewed literature with meta-analyses to quantitatively summarize the levels of tear cytokines in DED. Methods-The PubMed, Embase, Web of Science, Ovid, Cochrane, and Scopus databases were reviewed until September 2019, and original articles investigating tear cytokines in DED patients were included. Differences of cytokines levels of DED patients and controls were summarized by standardized mean differences (SMD) using a random effects model. Study quality was assessed by applying Newcastle-Ottawa-Scale and the GRADE quality score. Methods of analytical procedures were included as covariate. Results-Thirteen articles investigating 342 DED patients and 205 healthy controls were included in the meta-analysis. The overall methodological quality of these studies was moderate. Systematic review of the selected articles revealed that DED patients had higher tear levels of interleukin (IL)-1ß, IL-6, chemokine IL-8, IL-10, interferon-γ, IFN-γ, and tumor necrosis factor-α, TNF-α as compared to controls. Evidence was less strong for IL-2 and IL-17A. Conclusions-Data show that levels of tear cytokines in DED and control display a great variability, and further studies of higher quality enrolling a higher number of subjects are needed, to define a cut-off value.

In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in dry eye disease using newly developed fully automated system.

PURPOSE: To evaluate in vivo confocal microscopy (IVCM) features of corneal subbasal nerve plexus (SNP) in the setting of dry eye disease (DED) using fully automated software "ACCMetrics," and to further investigate its diagnostic performance in discriminating DED patients. METHODS: IVCM exams of SNP in DED patients and matched control subjects were performed using Heidelberg Retina Tomograph with the Rostock Cornea Module. The following parameters were obtained with ACCMetrics: corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal nerve total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW), and corneal nerve fractal dimension (CNFrD). The Mann-Whitney U test was used to compare variables. Receiver operating characteristic curves with calculations of the area under the curve (AUC) were used to describe the accuracy of IVCM parameters for discriminating DED patients from controls. RESULTS: Thirty-nine DED patients and 30 control subjects were included. Significantly, lower values of CNFD, CNBD, and CNFL and higher value of CNFW were found in DED patients compared to controls (respectively, 20.5 ± 8.7 vs 25.4 ± 6.7 n/mm2; 25.6 ± 20.1 vs 37.6 ± 21.5 n/mm2; 12.6 ± 4.4 vs 14.5 ± 2.9 mm/mm2; 0.021 ± 0.001 vs 0.019 ± 0.001 mm/mm2; always p < 0.024). CNFW value had the highest diagnostic power in discriminating DED patients (AUC = 0.828). When the diagnosis of DED was made based on either CNFW or CNBD, the sensitivity was 97.4% and the specificity 46.7%. CONCLUSIONS: The software ACCMetrics was able to rapidly detect SNP alterations occurring in the setting of DED and showed good diagnostic performance in discriminating DED patients.

Blood derived eye drops for the treatment of cornea and ocular surface diseases.

The use of blood derived eye drops for the treatment of ocular surface disorders has become increasingly popular in recent years. The mechanism of action is the stimulation of cellular proliferation and migration by supplying an active mixture of growth factors and cytokines at the ocular surface, thus mimicking the function of the lacking natural tears. Blood derived eye drops have been used in the last decades for the treatment of a variety of ocular surface diseases, including mainly dry eye disease, persistent corneal epithelial defect, corneal ulcer, ocular surface burn, recurrent corneal erosion and limbal stem-cell deficiency. Among overall blood derived eye drops, both autologous (from the patients themselves) and homologous (from donors) products exist, with different advantages and disadvantages. Autologous serum, obtained from the patient's own peripheral blood, is the first introduced and most commonly used product. Despite several randomized clinical trials showed its safety and efficacy, a recent Cochraine meta-analysis failed to show significant results due to low evidence. Homologous sources including allogeneic serum obtained from healthy donors, and umbilical cord blood serum collected at the time of delivery, are efficient alternatives, especially when autologous serum therapy is contraindicated or not appropriate. Platelet-derived eye drops are prepared and used in various but poor standardized preparations, namely platelet-rich plasma, plasma rich in growth factors, and platelet lysate. Future perspectives of blood-derived products include the introduction of tailored eye drops, screened for the proper content of growth factors and cytokines according to each patient and ocular surface disease.

Intolerant contact lens wearers exhibit ocular surface impairment despite 3 months wear discontinuation.

PURPOSE: To evaluate ocular surface (OS) parameters recovery in intolerant contact lens (CL) wearers after a period of discontinuation. METHODS: This is a retrospective analysis of data from 87 intolerant CL wearers who had discontinued their use for an average period of 12 weeks because of associated discomfort and failure to successfully refit. Data were collected from clinical charts. Data from 50 matched healthy volunteers served as controls. Clinical tests included subjective discomfort symptoms questionnaire (Ocular Surface Disease Index, OSDI), Schirmer test, break-up time (BUT), corneal esthesiometry and corneo-conjunctival staining. Laboratory tests included scraping and imprint cytology. Tear protein analysis included dosage of total tear protein (TP), lysozyme-C (LYS-C), lactoferrin (LACTO), zinc-α2-glycoprotein (ZAG-2), IgA heavy chain bands (Ig-A), and serum albumin (ALB). Data were correlated to wear parameters. RESULTS: All values were significantly worse in intolerant CL wearers group (p always <0.001). In particular, lower values compared to controls were found for BUT, corneal esthesiometry, goblet cell density, LYS-C, LACTO, ZAG-2, and TP. On the contrary, higher values compared to controls were found for OSDI, staining, imprint cytology, scraping cytology, ALB, IgA-heavy chain. The IgA/LYS-C ratio calculated as an index of the increased activity of the IgA-producing cell was found significantly higher in the intolerant group and in correlation with discomfort symptoms. CONCLUSIONS: Ocular surface parameters were altered in intolerant CL wearers, even after a prolonged discontinuation period. Our data suggest that OS recovery necessary to successfully refit lenses may need a discontinuation time longer than 3 months.

Development, Optimization, and Clinical Relevance of Lactoferrin Delivery Systems: A Focus on Ocular Delivery.

Lactoferrin (Lf), a multifunctional protein found abundantly in secretions, including tears, plays a crucial role in ocular health through its antimicrobial, immunoregulatory, anti-inflammatory, and antioxidant activities. Advanced delivery systems are desirable to fully leverage its therapeutic potential in treating ocular diseases. The process of Lf quantification for diagnostic purposes underscores the importance of developing reliable, cost-effective detection methods, ranging from conventional techniques to advanced nano-based sensors. Despite the ease and non-invasiveness of topical administration for ocular surface diseases, challenges such as rapid drug elimination necessitate innovations, such as Lf-loaded contact lenses and biodegradable polymeric nanocapsules, to enhance drug stability and bioavailability. Furthermore, overcoming ocular barriers for the treatment of posterior segment disease calls for nano-formulations. The scope of this review is to underline the advancements in nanotechnology-based Lf delivery methods, emphasizing the pivotal role of multidisciplinary approaches and cross-field strategies in improving ocular drug delivery and achieving better therapeutic outcomes for a wide spectrum of eye conditions.

Utilising Narrative Medicine to Identify Key Factors Affecting Quality of Life in Dry Eye Disease: An Italian Multicentre Study.

INTRODUCTION: Despite an improved understanding of its pathogenesis, dry eye disease (DED) remains relatively underestimated and its treatment challenging. A better alignment between the clinical evaluation and the patient self-assessment also requires capturing the whole patient experience of DED. This project aimed to unveil this experience through narrative medicine (NM). METHODS: The project involved 38 expert centres in Italy and one in San Marino, targeting adult patients with DED, their informal caregivers and their treating ophthalmologists. Written narratives and sociodemographic and quality of life (QoL)-related data were anonymously collected through the project's webpage. Narratives were analysed through MAXQDA (VERBI Software, Berlin, Germany), NM classifications and content analysis. RESULTS: A total of 171 patients with DED, 37 informal caregivers and 81 ophthalmologists participated in the research. DED was defined as a disabling condition by 19% of patients and 35% of caregivers; 70% of patients reported that a therapeutic alliance is an integral part of DED treatment and 32% hope for more effective therapies. Forty-four per cent of patients assessed their own QoL as good; however, DED emerged as importantly impacting work performance and social events. DED physical, emotional and economic burden and the cruciality of a trusting care relationship represent the main themes that emerged across all narratives, while empathy and effective treatment are among the factors favouring coping with DED. CONCLUSION: This project marked a pioneering initiative investigating the lived experience of patients with DED through NM, simultaneously involving all viewpoints involved in the care pathway. NM enabled the unveiling of factors favouring the ability to cope with DED and its associated QoL implications and provided valuable insights to improve the therapeutic alliance.

Diagnostic performance of a tear protein panel in early dry eye.

PURPOSE: To evaluate the tear protein pattern in patients with recent subjective symptoms of dry eye (DE) and with poor distinctive DE clinical signs. METHODS: One hundred sixty patients suspected of suffering from mild to moderate DE according to the Dry Eye Workshop (DEWS report 2007) severity grade and 45 matched normal volunteers were included in the study. Subjective symptom score (Ocular Surface Disease index score), Schirmer test I, tear film break-up time, cornea and conjunctiva staining (National Eye Institute score); and tear protein analysis were performed. Statistical evaluation of data was performed with Mann-Whitney unpaired and Student t tests, (significance p<0.05). Correlations between variables were evaluated by using Pearson's (r) or Spearman's (ρ) correlation coefficients. Thresholds were selected from receiver operating curves; sensitivity, specificity, likelihood ratio (LR+), and positive predictive values were calculated for each protein. The combination of variables was carried out by univariate analysis, representing the best combination of tests for early DE diagnosis. RESULTS: TOTAL PROTEIN CONTENT (TP) AND THE FOLLOWING PROTEINS WERE RECOGNIZED IN ALL SAMPLES: lysozyme-C (LYS-C), lactoferrin (LACTO), tear lipocalin 1 (LIPOC-1), zinc-alpha-2-glycoprotein (ZAG-2), transferrin (TRANSF), and exudated serum albumin (ALB). A statistically significant decrease was demonstrated between normal subjects and patients with DE (mg/ml, mean±SD) for TP (9.89±2.28 versus 6.44±2.1), LYS-C (3.06±1.07 versus 2.15±0.78), LIPOC-1 (1.71±0.52 versus 0.98±0.5), ZAG-2 (0.43±0.24 versus 0.25±0.2), TRANSF (0.9±0.6 versus 0.33±0.3), and LACTO (2.11±0.74 versus 1.47±0.76), while an increase was found for ALB (0.21±0.5 versus 0.94±1.28). LIPOC-1 and ZAG-2 were strongly correlated to tear film break-up time. The proteins were related to the DEWS severity grade. Changes in each protein were a better predictor of early DE than were clinical variables; TP, LIPOC-1, and ALB exhibited the highest diagnostic performance either alone (LR+ 16.7, 12.3, 4.7, respectively) or when combined in a univariate analysis (LR+: 41.8, positive predictive value: 99.9). CONCLUSIONS: Our results demonstrated in tears from patients with early DE a significant reduction in tear protein content as a whole, associated with a decrease in proteins with antibacterial and protective functions. A decrease in proteins with lipid binding properties and an increase in inflammatory-related proteins were also shown. Changes in the abundance of a panel of tear proteins with divergent functions was found to better diagnose early DE than did conventional clinical tests.

Astigmatism in patients with idiopathic congenital nystagmus.

PURPOSE: To evaluate the association between astigmatism and idiopathic congenital nystagmus (ICN) in infantile nystagmus syndrome (INS). MATERIALS AND METHODS: We analysed refractive errors in a cohort of 488 consecutive patients with ICN (group A) and further compared the results obtained with those of 488 age-matched controls with no nystagmus (group B). Only the worst eye was considered for statistical analysis. All patients were stratified into the following age groups: 1 to 4 years (age group 1); 5 to 12 years (age group 2); and 13 years to 57 years (age group 3) (mean age: 29). RESULTS: Three hundred and seventy patients (69.7 %) in group A and 269 patients (55,12 %) in group B had refractive errors. The types of refractive errors observed were: myopia, hyperopia (>0.50 dioptres) and astigmatism (>1.25 dioptres). Results in group A were as follows: 319 patients (65.37 %) were astigmatic, 34 (6.97 %) were hyperopic, and 17 (3.48 %) were myopic. Mean right-eye astigmatism was 2.72 dioptres, and mean left-eye astigmatism was 2.69 dioptres. Results in group B were as follows: 56 (11.47 %) were astigmatic, 165 (33.81) were hyperopic, and 48 (9.84) were myopic. Mean right-eye astigmatism was 2.05 dioptres, and mean left-eye astigmatism was 2.37 dioptres. The prevalence of astigmatism is greater, in the entire sample, for subjects from age groups 2 and 3 (p<0.005). It shows a tendency to increase with age for patients of group A and in age group 3 (p=0.009). CONCLUSIONS: Astigmatism is more common in patients with ICN than in the general population (65.37 % vs 11.47 %) (p<0.001). Astigmatism increases with age, with a very high statistical significance in patients 13 years old and above (age group 3) when nystagmus is also present. Thus, nystagmus appears to be a predisposing factor for both the presence of astigmatism and the development with the age of high values of this refractive error. This findings should be taken into due account when considering visual dysfunctions in nystagmic patients.

Human glial müller and umbilical vein endothelial cell coculture as an in vitro model to investigate retinal oxidative damage. A morphological and molecular assessment.

The aim of this study was to optimize a coculture in vitro model established between the human Müller glial cells and human umbilical vein endothelial cells, mimicking the inner blood-retinal barrier, and to explore its resistance to damage induced by oxidative stress. A spontaneously immortalized human Müller cell line MIO-M1 and human umbilical vein endothelial cells (HUVEC) were plated together at a density ratio 1:1 and maintained up to the 8th passage (p8). The MIO-M1/HUVECs p1 through p8 were treated with increasing concentrations (range 200-800 µM) of H2 O2 to evaluate oxidative stress induced damage and comparing data with single cell cultures. The following features were assayed p1 through p8: doubling time maintenance, cell viability using MTS assay, ultrastructure of cell-cell contacts, immunofluorescence for Vimentin and GFAP, molecular biology (q-PCR) for GFAP and CD31 mRNA. MIO-M1/HUVECs cocultures maintained distinct cell cytotype up to p8 as shown by flow cytometry analysis, without evidence of cross activation, displaying cell-cell tight junctions mimicking those found in human retina, only acquiring a slight resistance to oxidative stress induction over the passages. This MIO-M1/HUVECs coculture represents a simple, reproducible and affordable model for in vitro studies on oxidative stress-induced retinal damages.

Early Outcomes of an Artificial Endothelial Replacement Membrane Implantation After Failed Repeat Endothelial Keratoplasty.

PURPOSE: The purpose of this study was to report the outcomes of a novel artificial endothelial replacement membrane implant for treating corneal edema after failed repeat endothelial keratoplasty (EK). DESIGN: This was a retrospective interventional case series. METHODS: Patients with chronic corneal edema underwent removal of the EK graft and implantation of an artificial endothelial replacement membrane (EndoArt, EyeYon Medical, Israel) several months after 2 or more Descemet stripping endothelial keratoplasty procedures. The implant was secured to the posterior corneal surface using an air-gas bubble. Outcome measures included corrected distance visual acuity (logMAR), central corneal thickness, device-related complications, and ocular discomfort. RESULTS: Five eyes of 5 patients underwent EndoArt implantation. Six months after surgery, the synthetic endothelial replacement membrane was well-centered and adherent to the posterior corneal surface, with improvement in central corneal transparency in all patients. Corrected distance visual acuity increased from mean 1.26 ± 0.25 (logMAR) preoperatively to 0.74 ± 0.44 (logMAR) postoperatively (P = 0.06). Central corneal thickness significantly decreased from a mean of 805 ± 135 µm (excluding the EK graft) preoperatively to 588 ± 60 µm (excluding the EndoArt) postoperatively (P = 0.015). No severe device-related complications developed after surgery, although most patients required more than 1 air-gas bubble injection to achieve complete implant adhesion. All patients experienced preoperative reduction in subjective ocular pain. CONCLUSIONS: Synthetic endothelial replacement membrane implantation improves central corneal transparency and visual acuity in patients with failed EK and guarded prognosis for repeat keratoplasty. No significant implant-related adverse events occurred after surgery.

Longitudinal Changes of Ocular Surface Microbiome in Patients Undergoing Hemopoietic Stem Cell Transplant (HSCT).

PURPOSE: To evaluate changes in the ocular surface microbiome (OSM) between pre- and post-haemopoietic stem cell transplant (HSCT) in the same patient, and to assess the potential impact of these changes in ocular graft-versus-host disease (o)GVHD development. METHODS: Lower fornix conjunctival swabs of 24 patients were obtained before and after HSCT and subjected to DNA extraction for amplification and sequencing of the V3-V4 regions of the bacterial 16S rRNA gene. The obtained reads were reconstructed, filtered, and clustered into zero-radius operational taxonomic units (zOTUs) at 97% identity level before taxonomic assignment, and biodiversity indexes were calculated. Transplant characteristics were recorded, and dry eye was diagnosed and staged 1-4 according to the Dry Eye WorkShop (DEWS) score. RESULTS: No significant difference in OSM alpha diversity between pre- and post-transplant was found. A significant difference in beta diversity was observed between patients with a DEWS score of 1 versus 3 (p = 0.035). Increased corneal damage between pre- and post-HSCT was significantly associated with a decrease in alpha diversity. The changes in OSM were not associated with oGVHD, nor with any transplant parameter. CONCLUSIONS: This preliminary study is the first study to analyse changes in the OSM before and after HSCT longitudinally. No trend in OSM biodiversity, microbial profile, or overall composition changes before and after HSCT was significant or associated with oGVHD onset. The great variability in the observed OSM profiles seems to suggest the absence of a patient-specific OSM "signature".

TFOS Lifestyle: Impact of nutrition on the ocular surface.

Nutrients, required by human bodies to perform life-sustaining functions, are obtained from the diet. They are broadly classified into macronutrients (carbohydrates, lipids, and proteins), micronutrients (vitamins and minerals) and water. All nutrients serve as a source of energy, provide structural support to the body and/or regulate the chemical processes of the body. Food and drinks also consist of non-nutrients that may be beneficial (e.g., antioxidants) or harmful (e.g., dyes or preservatives added to processed foods) to the body and the ocular surface. There is also a complex interplay between systemic disorders and an individual's nutritional status. Changes in the gut microbiome may lead to alterations at the ocular surface. Poor nutrition may exacerbate select systemic conditions. Similarly, certain systemic conditions may affect the uptake, processing and distribution of nutrients by the body. These disorders may lead to deficiencies in micro- and macro-nutrients that are important in maintaining ocular surface health. Medications used to treat these conditions may also cause ocular surface changes. The prevalence of nutrition-related chronic diseases is climbing worldwide. This report sought to review the evidence supporting the impact of nutrition on the ocular surface, either directly or as a consequence of the chronic diseases that result. To address a key question, a systematic review investigated the effects of intentional food restriction on ocular surface health; of the 25 included studies, most investigated Ramadan fasting (56%), followed by bariatric surgery (16%), anorexia nervosa (16%), but none were judged to be of high quality, with no randomized-controlled trials.

A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins.

PURPOSE: To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method. METHODS: A total of 5 µl of tears were collected from 25 patients diagnosed as having mild to moderate dry eye according to Dry Eye Workshop guidelines and from 20 matched normal volunteers. Protein analysis was performed with the 2100 Bioanalyzer; different protein kit assays were evaluated (Protein 80 kit, Protein 230 kit, High Sensitivity Protein 250 kit) for sizing and quantifying protein samples from 5 to 80 kDa, 14 to 230 kDa, and 5 to 250 kDa, respectively. A standard protein ladder was loaded on each chip to allow an estimation of the appropriate molecular weight of the separated proteins; a sample buffer containing a lower and an upper marker was used to check the correct alignment of each lane. Virtual bands generated by the Bioanalyzer were identified and validated as follows: tear samples were run in parallel and proteins separated by one-dimensional and two-dimensional sodium dodecyl sulfate-PAGE and characterized by immunoblotting, enzymatic digestion, and analysis with liquid chromatography-mass spectrometry followed by a search of the SProt human protein database. RESULTS: Analyses were successfully performed by using as small as a 2 µl tear sample. The Protein 230 kit was selected as the best chip kit, able to differentiate all the proteins of interest. The measurement noise parameters were low, and reproducibility and repeatability exhibited high accuracy (0.998 and 0.995, respectively) and precision (0.974 and 0.977, respectively). The coefficient of variability was slightly higher than that declared by the manufacturer (6.2% versus 5.0%). Total protein content and the following proteins were recognized in all samples: lipophilin A lysozyme C, tear lipocalin-1, zinc-alpha-2-glycoprotein, serotransferrin, lactotransferrin, and exudated serum albumin. CONCLUSIONS: Our data demonstrate that this chip-based tear protein analysis is a reliable method of instrumental diagnosis in daily clinical activity and may provide supporting evaluation parameters for diagnosing and managing tear-based disorders.

Post Penetrating Keratoplasty Ectasia: Incidence, Risk Factors, Clinical Features, and Treatment Options.

BACKGROUND: Corneal transplantation in keratoconus (KC) patients is generally considered to be successful with a high grade of patient satisfaction. Long-term studies suggest a 6% to 11% probability of KC recurrence manifested by keratometric instability and progressive corneal ectasia. METHODS: We propose to review the frequency, risk factors for the development, and the surgical options for the correction of high irregular astigmatism due to late graft ectasia following penetrating keratoplasty (PK). RESULTS: Post-keratoplasty ectasia is characterized by increasing corneal steepening with myopic shift and high irregular astigmatism, developing years or decades after PK, mostly occurring in KC patients. Contact lenses may adequately improve the visual acuity; however, because these patients are often elderly and intolerant to hard contact lenses, ultimately a surgical correction is proposed to the patient. Compressive suture and corneal wedge resection may improve corneal astigmatism, but the outcomes are unpredictable and often temporary. For this reason, a larger PK graft is often proposed for surgical rehabilitation with the consequence of removing more of the recipient's healthy endothelium and exposing the patient to a renewed immunogenic stimulus and short-term graft failure for endothelial decompensation. More recently, lamellar keratoplasty using various techniques has been proposed as an alternative to PK in order to maximize the visual outcomes and minimize the complications. CONCLUSIONS: Management of advanced corneal ectasia is a significant challenge for corneal surgeons. Many surgical approaches have been developed, so there is a large arsenal of surgical operations to correct post-PK ectasia. Among them, large-diameter anterior lamellar keratoplasty may be a viable, safer, and effective alternative to PK for the correction of post-keratoplasty ectasia.

Comparison of Trehalose/Hyaluronic Acid (HA) vs. 0.001% Hydrocortisone/HA Eyedrops on Signs and Inflammatory Markers in a Desiccating Model of Dry Eye Disease (DED).

BACKGROUND: Dry eye disease (DED) is a multifactorial disease where ocular surface inflammation and damage play key etiological roles. PURPOSE: To compare a combination of 3% trehalose (T) and 0.15% hyaluronic acid (HA) (Thealoz duo®, T/HA) with a tear substitute containing 0.001% hydrocortisone (I) and 0.2% HA (Idroflog®, I/HA), with respect to changes on signs and inflammatory markers in a mouse DED model. METHODS: Thirty 12-week-old C57BL/6 mice were exposed in a controlled-environment chamber as a desiccating stress model of DED for 35 days. At day 14 (T1), administration of 5 µL T or I in the right eye (RE) or NaCl 0.9% in the left eye (LE) started, twice a day. Animals were sacrificed after 7 (T2), 14 (T3), 21 (T4, endpoint) days from the beginning of treatment. Corneal fluorescein staining ratio (Image J), histological and histochemical assessment of ocular surface tissues (goblet cell GC density and characterization -PAS, Alcian blue pH 2.5, pH 1.0, and MUC4 expression-in the superior and inferior conjunctiva), and levels of inflammatory markers HLA-DR, IL-1ß and TNF-α in cornea and conjunctiva were measured. RESULTS: No animal fully recovered from DED signs at the endpoint. Difference between arms was observed at T3 and T4, with T treated eyes showing a higher corneal damage reduction, PAS-positive GC recovery, lower inflammatory marker expression as compared to the I treated ones. CONCLUSIONS: Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA. Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA.

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing.

BACKGROUND: We evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability. MATERIALS AND METHODS: Twenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment. RESULTS: All the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources. DISCUSSION: Protocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.